Diphtheria is the prototype of an infectious disease mediated by a bacterium, corynebacterium diphtheriae, which produces a potent exotoxin. Intoxication of sensitive cells by diphtheria toxin involves: 1) binding of the toxin, through its B-fragment, to specific membrane receptors on the cell surface, 2) processing and translocation of the A-fragment through the plasma membrane into the cytoplasm, and 3) inhibition of protein synthesis by ADP-ribosylation of elongation factor 2, a reaction catalyzed by the A-fragment. The objective of this research proposal is directed towards elucidation of the biology and biochemistry of diphtheria toxin receptors (point 1 above). We have demonstrated the presence of specific diphtheria toxin binding membrane glycoproteins from the surface of cells from guinea pigs and hamsters, two toxin sensitive species. The method for detection of these glycoproteins consists of preparation of a glycoprotein enriched fraction from exogenously radioiodinated cells, followed by immunoprecipitation with diphtheria toxin plus antiserum to diphtheria toxin. We propose to purify these diphtheria toxin-binding glycoproteins, to produce specific antibodies to them, and to utilize these antibodies to determine if the toxin-bindng glycoproteins are physiological revelant receptors. The possible presence of similar diphtheria toxin binding molecules on the surface of cells from mouse and rat, two toxin resistant species, will also be investigated. The biochemical nature of the diphtheria toxin binding site on the cell surface glycoproteins from toxin sensitive cells will be investigated. In addition, the receptor binding domain on the B-fragment of the toxin molecule will be identified, purified, tested for its ability to protect sensitive cells from the action of toxin, and its primary sequence determined. The results of this proposed research will significantly extend our current knowledge concerning the fundamental mechanism utilized by cells for the receptor mediated internalization of biologically active macromolecules such as toxins and hormones.